Studies on the aetiology of kiwifruit decline: interaction between soil-borne pathogens and waterlogging

Aims: In 2012, Italian kiwifruit orchards were hit by a serious root disease of unknown aetiology (kiwifruit decline, KD) that still causes extensive damage to the sector. While waterlogging was soon observed to be associated with its outbreak, the putative role of soil microbiota remains unknown. This work investigates the role of these two factors in the onset of the disease. Methods: Historical rainfall data were analysed to identify changes that might explain KD outbreak and mimic the flooding conditions required to reproduce the disease in a controlled environment. A greenhouse experiment was thus designed, and vines were grown in either unsterilized (U) or sterilized (S) soil collected from KD-affected orchards, and subjected (F) or not (N) to artificial flooding. Treatments were compared in terms of mortality rate, growth, and tissue modifications. Results: KD symptoms were only displayed by FU-treated vines, with an incidence of 90%. Ultrastructural observations detected tyloses and fibrils in the xylem vessels of all plants, irrespective of the treatment. Phytopythium vexans and Phytopythium chamaehyphon, isolated from roots of FU plants, emerged as the associated microorganisms. Conclusions: We succeeded in reproducing KD under controlled conditions and confirmed its association with both waterlogging and soil-borne microorganism(s)

A Prevention through Design Approach for the Environmental S&H Conditions and the Ventilation System at an Italian Underground Quarry

Even if the number of the Italian quarrying operations in underground is steadily growing, in many cases the safety criticalities are somehow underestimated, in spite of the regulations D.Lgs. 81/08 and D.Lgs. 624/96, Italian enforcements of the European Directives. Ventilation system is conceived to face very simplified requirements, whilst critical pollutants or emergency situations are not taken in due considerations. Asides, the ventilation system fault and availability analysis is seldom included in the project. The paper deals with the results of a research work started some years ago at an underground quarry exploited through drift sublevel based on drill and blast technique, to identify criteria suitable to grant effective safety and health -S&H- conditions for the workers operating in the underground in the Prevention through Design approach. Taken into account that the phases for an effective approach to the S&H problems in underground must follow a hierarchic method in which the risk management should be faced from an effective hazard reduction to a minimum at the sources, and the ventilation should be considered only as a 4th level solution, the possibilities of control at the main pollution sources, i.e. the emission of pollutants due to the rock winning and mucking operations, have been examined. The residual risk was then faced with both the original underground and airways layout definition for a new exploitation development, based on technical and efficiency considerations, and on fire emergency computer simulations. Finally, the paper summarizes the results of an availability analysis of the ventilation system for the normal operating conditions and the emergencies management, on the basis of the results of Hazard Evaluation techniques, in particular Hazard and Operability Analysis and Fault Tree Analysis

Determinismo genético e molecular do metabolismo de diterpenos em Coffea spp.

Cafestol e caveol são os dois principais diterpenos presentes nos frutos de café. Esses compostos específicos do cafeeiro têm se mostrado importantes na saúde humana, induzindo alterações no colesterol e ações anti-cancerígenas. Apesar da sua importância, há pouca informação sobre os princípios genéticos e moleculares de seu metabolismo. Análises fenotípicas através de HPLC, com cafés de diferentes espécies (vários genótipos por espécie), indicam uma variabilidade importante para cafestol, caveol e 16OMC. As análises in silico dos EST de Coffea permitiram identificar cDNAs parciais correspondente a um gene de CPS, dois de KO e um de KS. Análises de expressão desses genes por RTq-PCR quantitativa, em tecidos separados durante o desenvolvimento dos frutos, estão em andamento. Resultados preliminares indicam que os quatro genes alvos apresentam expressão diferencial durante o desenvolvimento dos tecidos do fruto. Os resultados de expressão serão discutidos considerando o interesse na identificação dos genes potencialmente envolvidos na regulação da concentração de cafestol e caveol

Recombinase mediated cassette exchange into genomic targets using an adenovirus vector

Recombinase mediated cassette exchange (RMCE) is a process in which site-specific recombinases exchange one gene cassette flanked by a pair of incompatible target sites for another cassette flanked by an identical pair of sites. Typically one cassette is present in the host genome, whereas the other gene cassette is introduced into the host cell by chemical or biological means. We show here that the frequency of cassette exchange is dependent on the relative and absolute quantities of the transgene cassette and the recombinase. We were able to successfully modify genomic targets not only by electroporation or chemically mediated gene transfer but also by using an adenovirus vector carrying both the transgene cassette to be inserted and the recombinase coding region. RMCE proceeds efficiently in cells in which the adenovirus vector is able to replicate. In contrast, insufficient quantities of the transgene cassette are produced in cells in which the virus cannot replicate. Additional transfection of the transgene cassette significantly enhances the RMCE frequency. This demonstrates that an RMCE system in the context of a viral vector allows the site directed insertion of a transgene into a defined genomic site

Microparticle-mediated transfer of the viral receptors CAR and CD46, and the CFTR channel in a CHO cell model confers new functions to target cells

Cell microparticles (MPs) released in the extracellular milieu can embark plasma membrane and intracellular components which are specific of their cellular origin, and transfer them to target cells. The MP-mediated, cell-to-cell transfer of three human membrane glycoproteins of different degrees of complexity was investigated in the present study, using a CHO cell model system. We first tested the delivery of CAR and CD46, two monospanins which act as adenovirus receptors, to target CHO cells. CHO cells lack CAR and CD46, high affinity receptors for human adenovirus serotype 5 (HAdV5), and serotype 35 (HAdV35), respectively. We found that MPs derived from CHO cells (MP-donor cells) constitutively expressing CAR (MP-CAR) or CD46 (MP-CD46) were able to transfer CAR and CD46 to target CHO cells, and conferred selective permissiveness to HAdV5 and HAdV35. In addition, target CHO cells incubated with MP-CD46 acquired the CD46-associated function in complement regulation. We also explored the MP-mediated delivery of a dodecaspanin membrane glycoprotein, the CFTR to target CHO cells. CFTR functions as a chloride channel in human cells and is implicated in the genetic disease cystic fibrosis. Target CHO cells incubated with MPs produced by CHO cells constitutively expressing GFP-tagged CFTR (MP-GFP-CFTR) were found to gain a new cellular function, the chloride channel activity associated to CFTR. Time-course analysis of the appearance of GFP-CFTR in target cells suggested that MPs could achieve the delivery of CFTR to target cells via two mechanisms: the transfer of mature, membrane-inserted CFTR glycoprotein, and the transfer of CFTR-encoding mRNA. These results confirmed that cell-derived MPs represent a new class of promising therapeutic vehicles for the delivery of bioactive macromolecules, proteins or mRNAs, the latter exerting the desired therapeutic effect in target cells via de novo synthesis of their encoded proteins

Syndecan 4 Is Involved in Mediating HCV Entry through Interaction with Lipoviral Particle-Associated Apolipoprotein E

Hepatitis C virus (HCV) is a major cause of liver disease worldwide and HCV infection represents a major health problem. HCV associates with host lipoproteins forming host/viral hybrid complexes termed lipoviral particles. Apolipoprotein E (apoE) is a lipoprotein component that interacts with heparan sulfate proteoglycans (HSPG) to mediate hepatic lipoprotein uptake, and may likewise mediate HCV entry. We sought to define the functional regions of apoE with an aim to identify critical apoE binding partners involved in HCV infection. Using adenoviral vectors and siRNA to modulate apoE expression we show a direct correlation of apoE expression and HCV infectivity, whereas no correlation exists with viral protein expression. Mutating the HSPG binding domain (HSPG-BD) of apoE revealed key residues that are critical for mediating HCV infection. Furthermore, a novel synthetic peptide that mimics apoE's HSPG-BD directly and competitively inhibits HCV infection. Genetic knockdown of the HSPG proteins syndecan (SDC) 1 and 4 revealed that SDC4 principally mediates HCV entry. Our data demonstrate that HCV uses apoE-SDC4 interactions to enter hepatoma cells and establish infection. Targeting apoE-SDC interactions could be an alternative strategy for blocking HCV entry, a critical step in maintaining chronic HCV infection